G protein-coupled receptors (GPCRs) signal through 2 major pathways via small G proteins and β-arrestins, triggering downstream cell signaling cascades. The physiological impact of the cell signaling effector functions affect human health and disease pathologies. The bifurcation of GPCR signal transduction between G proteins and arrestins has attracted major interest in academic and pharmaceutical research on target-related pathogenesis in recent years. More importantly, possibilities of designing compounds that preferentially activate or inhibit specific GPCR signal transduction pathways have brought new hope for finding efficacious therapies without harmful side effects to the drug discovery community.
There have been a number of established technologies used to measure GPCR β-arrestin signaling. These older β-Arrestin screening assays that can be used in high throughput screening all require tagging GPCRs intracellularly at the C-terminus with an enzyme, enzyme fragment, or fluorescence protein. As C-terminus of GPCRs are the functional domains that bind to G proteins and arrestins, these sizable protein tags can themselves cause biases in receptor signaling by altering protein-protein interactions, confounding the analysis of experimental results. Furthermore, some studies resort to using one cell line with unmodified GPCR for G protein pathway analysis and tagged receptor in another cell line for arrestin analysis. This approach further introduces unwanted biases generated from different cell lines, which are irrelevant to the target GPCR-triggered cell signaling. Finally, the requirement of GPCR tagging from the older β-arrestin screening assays makes studies of endogenous GPCRs completely unattainable. The need to overcome these limitations has led to the genesis of Multispan’s MULTISCREEN™ β-Arrestin Sensor Technology, specifically designed to measure β-Arrestin recruitment by unmodified GPCRs.
The MULTISCREEN™ β-Arrestin Sensor Technology employs a bicistronic mammalian expression vector that carries 2 fusion proteins:
The luciferase enzyme fragments are proprietary whereas the membrane anchor protein is ubiquitously expressed on the cellular plasma membrane. This bicistronic insert has also been cloned into a MacMam viral expression vector for robust transient expression of the β-Arrestin sensor in a variety of cell lines.
In cells co-expressing the MULTISCREEN™ β-Arrestin Sensor and an endogenous or exogenous unmodified GPCR target, the activation of GPCR by its agonists leads to the recruitment of arrestins to the GPCR on the cell membrane. The proximity of the two fusion proteins (β-Arrestin-NLuc; Membrane-Anchor-CLuc) lead to the reconstitution of the whole luciferase enzyme activity through complementation, which can be detected by a luminescence substrate. The signal can be read conveniently by standard microplate readers.
Using this technology, we were able to generate HTS assay-ready stable cell lines for a variety of GPCRs. For example, we developed stable cell lines for all 4 opioid receptors. Specifically, the MULTISCREEN™ β-Arrestin Sensor expressed in MOR stable cell line clearly differentiated efficacies of super agonist DAMGO, full agonist Morphine and partial agonist Buprenorphine. In addition, the MOR β-arrestin recruitment assay gave >0.7 Z’ for high quality high throughput screening.
Leveraging the differences between β-arrestin and cAMP signaling kinetics and assay readouts, we developed high throughput and multiplexed screening methods for both assays in the same cell line and the same well. The dose-response curves from MULTISCREEN™ MOR stable cell line below were generated from such multiplexed assay format: β-arrestin luminescence assay was read first followed by TR-FRET cAMP assay as the 2nd readout.
“Two Assays in One Well” provides added advantage to the MULTISCREEN™ β-Arrestin Sensor Technology in signaling bias compound profiling by eliminating the one more well-to-well variable. In addition to high quality data, while “Two Assays in One Well” also provides 50% reduction of time and cost of materials related to cells, media, plates and supplies.
Besides using MULTISCREEN™ stable cell lines co-expressing GPCR and β-arrestin, we developed proprietary BacMam viral expression vector that enables robust transient expression of the β-arrestin sensor in recombinant or native cells. It proves to be simpler, less expensive, faster, and gentler than the various traditional transient transfection methods. By way of example, MULTISCREEN™ MRGX2 Stable Cell Line was transduced with β-Arrestin1 and β-Arrestin2 BacMam viral particles and assayed robustly in the MULTISCREEN™ β-Arrestin recruitment assay 48 hours later.
The innovation of MULTISCREEN™ β-Arrestin Sensor and diverse methods of use brought to the drug discovery research community a new way of assaying signal transduction through β-arrestins without having to tag GPCRs. This high throughput screening technology can be used in MULTISCREEN™ Stable Cell Lines that co-express GPCRs and β-Arrestin, and transiently introduced with BacMam into primary or heterologous cells expressing unmodified GPCRs stably or transiently.